Ototoxicants produce hearing loss by disrupting cochlear function, but mechanisms underlying the toxicities of the structurally diverse agents which damage the cochlea are elusive. Many ototoxicants generate reactive oxygen species (ROS) in organs outside of the cochlea, and ROS have been implicated in the hearing loss due to several pharmaceutical ototoxicants. However, prior to the original submission of this proposal, ROS formation had never been directly examined in the cochlea, nor had the effects of direct ROS generation within the cochlea and the role that ROS generation plays in environmental chemical ototoxicity been studied. We hypothesize that ROS generation is one of the mechanisms by which many ototoxicants, including trimethyltin (TMT), damage the cochlea and induce hearing loss. Specifically, we aim to: 1) determine whether ROS generation within the cochlea leads to similar disrupted function as is seen following TMT administration, by recording cochlear potentials (cochlear microphonic (CM), compound action potential (CAP), endocochlear potential (EP)) after intracochlear infusion of ROS generating systems, assessing the ability of ROS scavengers to protect against cochlear disruption and by correlating these changes with assayed levels of malondialdehyde (MDA), conjugated dienes and carbonyl in the cochlea, 2) determine, in vivo and in vitro, whether TMT exposure results in O-(2) and OH. production detectable by electron paramagnetic resonance spectroscopy (EPR) and H2O2 formation using the PeroXOquant assay, and whether such levels correlate with MDA, conjugated diene and carbonyl production, 3) determine whether ROS formation is involved in the acute disruption of CAP, and 4) later disruption of CM, which follow TMT administration, using cochlear electrophysiology, ROS scavengers and glutathione depleting agents at two distinct time periods following TMT administration, and separately correlating these two functional effects with levels of lipid peroxidation and protein oxidation at the times of functional loss, and 5) determine the ROS related effects of TMT on its primary cochlear target, the outer hair cell, in terms of cell morphology, subcellular peroxide and carbonyl production, and the induction of SODs along the basilar membrane. New data from our laboratory provide strong support for a role of ROS in TMT induced ototoxicity. Showing that ROS are formed in the cochlea following environmental toxicant exposure, along with relating subsequent levels of biochemical damage with cochlear dysfunction when ROS levels are altered, will aid in the understanding of ROS related cochlear disruption, and the prediction of the ototoxic potential of environmental chemicals, and may lead to clinically relevant methods of protecting against ototoxicity.